Pan-antiviral peptides for protein kinase inhibition

ABSTRACT

A method of inhibiting protein kinases by administering polypeptides derived from alpha-neurotoxin, and inhibiting protein kinases. Diseases treated thereby include cancer, influenza, Tourette&#39;s syndrome, pain, and neurological deficits.

CROSS-RELATED REFERENCE SECTION

This application is a divisional application of U.S. patent applicationSer. No. 12/691,902, filed Jan. 22, 2010, which is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The present invention relates to inhibition of protein kinases. Inparticular, the present invention relates to therapeutic uses ofpolypeptides that inhibit protein kinases.

DESCRIPTION OF RELATED ART

The inventions described herein arose from the pioneering work ofSanders, which documented the antipolio action of detoxified venoms fromElapidae. His work was preceded by the original observations of Bodianand Howe (Bull. Johns Hopkins Hospital, 69: 79-85 (1941)) that provedand measured the retrograde axonal transport of polio viruses to thecentral nervous system. The latter phenomenon was accomplished byseverance of the sciatic nerve in rats at different times followingviral infection of the footpads distal to the severed nerves. Sanderswas also cognizant of the early studies, by Lamb and Hunter (Lancet, 1:20-22 (1904)), of the pathology of cobra bites in patients in India, andin experimental animals injected with those venoms. Changes instructures within higher centers of the CNS, including centralchromatolysis, occurred within 1-4 hours following cobra envenimation.Further, it was long recognized from clinical observations that rabiesviruses, and proteins such as tetanus toxin (Wright et al., Brit. J.Expl. Path. 32: 169 (1951)), appeared to travel to the central nervoussystem via nerve pathways. From those and other observations Sandersinitiated his extensive studies into the ability of neurotoxic snakevenoms, detoxified by a specific chemical means, to prevent polioinfections in mice, rats and monkeys challenged with polioviruspreparations (Sanders, et al., Ann. N.Y. Acad. Sci. 58: 1-12 (1953)).Those studies were based on what was known at the time as the“interference” phenomenon in which infection with one virus conveysresistance to a second virus acquired a short time later.

With the availability of the Salk vaccine in the early 1950's Sanderspolio work was discontinued. He then initiated studies into effects ofhis medicines on the progressive, irreversible neuromusculardysfunctions in patients with amyotrophic lateral sclerosis (ALS). Hisclinical studies employed a Time-Series protocol, the statistical meanswith which to evaluate drug effects in patients such as those with ALS.By that means he circumvented the ethical problem of placeboadministration to patients with a progressive, irreversible disease.

The realization that protein toxins bind strongly to specific receptorson target cells surged with the discovery, by van Heyningen and Miller(J. Gen. Microbiol., 24: 107 (1961)), that tetanus toxin binds stronglyto gangliosides, chemical constituents of nerve cells. That phenomenoninitiated wide-ranging investigations by biochemists into the strongaffinities many protein toxins exhibit toward their specific receptorson cells. Further, they identified nontoxic fragments from within therespective protein toxins that retained the cell-binding functions. Suchnontoxic, cell-binding fragments offer potential therapeutic anddiagnostic opportunities, the goals of many subsequent studies. In 1977Miller et al., employing separations technology, identified thealpha-neurotoxins in the venoms as precursors of the active principalsin Sander's medicine (Biochim. Biophys. Acta, 496: 192-196 (1977)).Exploitation of the phenomenon quickly followed. The amelioration ofherpes virus infections in tissue culture systems and in experimentalanimals was defined by Yourist et al. (J. Gen. Virol., 64: 1475-1481(1983)). Lentz et al., (Biochem., 30: 10949-10957 (1991)) identifiedconstituents on rabies viruses that have amino acid sequences homologouswith those in the alpha-neurotoxins. Similar structures also occur inthe human immunodeficiency viruses (Bracci et al., Arch. Virol., 114:265-268 (1990)), also a neurotropic virus.

The ability of the nontoxic peptide preparations to inhibit neurotropicviruses supported Sander's original hypothesis that neurotoxic venomconstituents retain affinities for receptor sites on cells, providingone mechanism for the cell protection from those viruses. The surprisingdiscovery that the same nontoxic derivatives of the animal neurotoxinsalso inhibit the neuraminidases of a number of myxoviruses (Miller andAustin, U.S. Pat. No. 7,259,237, herein incorporated by reference)assigns a second antiviral property to the same peptides. Viralneuraminidases are required for the release of newly formed myxovirusvarions from their sites of origin.

The present invention now adds a third mechanism by which the samenontoxic toxin-derived peptides modulate biologic phenomena. Theyinhibit protein kinases such as those in heart muscle and human myelinas described below.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of inhibiting protein kinases,including the steps of administering polypeptides derived fromalpha-neurotoxin, and inhibiting protein kinases.

The present invention also provides for treatments of cancer, influenza,Tourette's syndrome, pain, and neurological deficits by the abovemethod.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 is a graph displaying the inhibition of the catalytic subunit ofpurified heart protein kinase A by an oxidized form of the antiviralpeptides;

FIG. 2 is a graph that provides a Lineweaver-Burke analysis of effectsof varying substrate concentrations on the inhibition of the heartprotein kinase A by three concentrations of the oxidized peptide; and

FIG. 3 is a graph that documents inhibition of the cyclic adenosinemonophosphate (cAMP) independent protein kinases of purified humanmyelin, wherein the reaction mixtures are the same as those describedfor FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally provides for a method of inhibitingprotein kinases, including the steps of administering polypeptidesderived from alpha-neurotoxin, and inhibiting protein kinases.

The polypeptides of the present invention are prepared as described inU.S. Pat. No. 7,259,237 to Applicants. Briefly, alpha-neurotoxin, aprecursor molecule, is modified either by specific oxidation of thedisulfide bonds, or by specific reduction and subsequent alkylation ofthe disulfide bonds. Various other modifications can be made to thepolypeptides as described in the above referenced patent. Thepolypeptides are also referred to as PANAVIRA® throughout theapplication.

Protein kinases are enzymes that modify proteins (in general serine,threonine, and/or tyrosine, among other amino acids) by chemicallyadding phosphate groups to the proteins. Protein kinases regulate themajority of cellular pathways and signal transduction methods. Theactivity of protein kinases is highly regulated because they have suchimportant effects on cells. Abnormal regulation, i.e. overactivity, ofprotein kinases often leads to disease. Therefore, it is of interest inthe present invention to regulate the function of protein kinases byinhibition.

The ability of the polypeptides to inhibit protein kinases, shown forthe first time herein, is one of three mechanisms of action that theypossess. Data below shows that protein kinases are inhibited in theheart and myelin. When administered, however, the polypeptides can alsofunction to inhibit viral neuraminidase, cause neurotropism, andcombinations thereof. Different mechanisms of action can be required intreating different diseases. These are further detailed below in theexamples.

Preferably, the polypeptides are administered by injection once daily tothe patient in need thereof. The first dose can be greater (i.e. bolusdose) or less than subsequent doses. In general, 0.2 ml to 0.4 ml of thepolypeptides are administered. Further administration methods aredescribed below.

The polypeptides can be used in a method of treating cancer. Morespecifically, the polypeptides are administered and protein kinases areinhibited. Kinases are commonly activated in cancer cells, such asc-Src, c-Abl, mitogen activated protein (MAP) kinase,phosphotidylinositol-3-kinase (PI3K) AKT, and the epidermal growthfactor (EGF) receptor. These kinases are known to contribute totumorigenesis. Activation of many of the kinases occurs in the samesignaling pathway. For example, HER-kinase family members (HER1[EGFR],HER3, and HER4) transmit signals through MAP kinase and PI3 kinase topromote cell proliferation. The polypeptides are shown to be effectivein treating malignant melanoma in the examples below. In malignantmelanoma, the polypeptides cause neurotropism as well as inhibition ofprotein kinases. MAP kinase is down-regulated and serine kinase isinhibited, causing the down-regulation of production of vascularendothelial growth factor (VEGF) and tissue factor (TF). Thepolypeptides can be used for treating cancers such as, but not limitedto, bladder cancer, breast cancer, colon and rectal cancel, endometrialcancer, kidney cancer, leukemia, lung cancer, melanoma, non-HodgkinLymphoma, pancreatic cancer, prostate cancer, skin cancer, and thyroidcancer.

The polypeptides can also be used in a method of treating influenza.Besides inhibiting protein kinases, the polypeptides inhibitneuraminidases when treating influenza. Many different types ofinfluenza can be treated, such as, but not limited to, influenza type A,influenza type B, and influenza type C. The influenza can be canineinfluenza as described in the examples below, and in this case, theneuraminidases inhibited are constituents of myxoviruses.

The polypeptides can be used in a method of treating Tourette'ssyndrome. Tourette's syndrome is generally characterized by the presenceof multiple physical tics and/or vocal tics and speech impediments.Common tics include eye blinking, coughing, throat clearing, sniffing,and facial movements. By inhibiting protein kinases, and morespecifically by modulating protein kinase responses to nerve cellstimuli, the polypeptides treat and remove these symptoms.

The polypeptides are also used in a method of alleviating pain, i.e. asan analgesic. The pain can be caused by many different conditions, suchas, but not limited to, cancer, neurologic pain, neurological deficits,stroke, chronic fatigue syndrome, and fibromyalgia. By alleviating painthrough neurotropism and protein kinase inhibition, the polypeptidesallow individuals with these diseases to function in day to dayactivities such as walking and various motor movements that theypreviously were unable to do because of pain.

The polypeptides can also be used in a method of overcoming neurologicaldeficits by inhibiting protein kinases. The neurological deficits thatare overcome can be any condition that affects nerve function such asneuron communication. Symptoms of neurological deficits include, but arenot limited to, weakness, paralysis, impaired hearing or vision, loss ordisturbance of sensation, impairment or loss of speech, fixed Dystonia,tremor, Myoclonus, other movement disorders, and Gait problems. Thepolypeptides can be used to overcome these symptoms. For example, thepolypeptides can be used to overcome paralysis. The inhibition ofprotein kinases can promote recovery of function after paralysis, asshown in the examples below.

The compounds of the present invention are administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the compounds of the presentinvention can be administered in various ways. It should be noted thatthey can be administered as the compound and can be administered aloneor as an active ingredient in combination with pharmaceuticallyacceptable carriers, diluents, adjuvants and vehicles. The compounds canbe administered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally,intratonsillar, and intranasal administration as well as intrathecal andinfusion techniques. Implants of the compounds are also useful. Thepatient being treated is a warm-blooded animal and, in particular,mammals including man. The pharmaceutically acceptable carriers,diluents, adjuvants and vehicles as well as implant carriers generallyrefer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

The doses can be single doses or multiple doses over a period of severaldays. The treatment generally has a length proportional to the length ofthe disease process and drug effectiveness and the patient species beingtreated.

When administering the compounds of the present invention parenterally,they will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include: U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the present invention should in no way beconstrued as being limited to the following examples, but rather, beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

Example 1 Protein Kinase Inhibition

Searches continued for additional mechanisms by which to explain therange of antiviral actions and other physiologic and clinical effectsdescribed in this document. As described in the introduction above boththe whole cobra venom and its purified alpha-neurotoxin, detoxifiedchemically, blocked infections with a variety of neurotropic viruses.Those viruses included polio viruses (Sanders, et al., Ann. N.Y. Acad.Sci. 58: 1-12 (1953)), Semliki Forest Virus (Miller, et al., Biochim.Biophys. Acta, 496: 192-196 (1975)), and herpes viruses (Yourist,Miller, et al., J. Gen. Virol., 64: 1475-1481 (1983)). Alpha-neurotoxinsare well known to bind strongly to acetylcholine receptors at myoneuraljunctions. Also well known is the presence in acetylcholine receptors ofa 65 Kd polypeptide that serves as a phosphorylation substrate forprotein kinases, a process that is inhibited by cholinergic ligands(Gordon, et al. Nature (1977) 267: 539-540: Teichberg, et al., Nature(1977) 267: 540-542). Since protein kinases mediate cellular responsesto events both at the surface and within cells (“signal reactions”)investigations centered on effects the nontoxic neurotoxin derivativeshave in modulation of protein kinases.

The following systems demonstrate inhibition of both cyclic AMP(cAMP)-dependent and cAMP-independent protein kinases by the antiviralpeptides. Reaction mixtures contained varying concentrations of eitherthe purified cobra alpha-neurotoxin or its oxidized, or reduced,alkylated derivatives prepared as described in U.S. Pat. No. 7,259,237.Protein kinase A sources in these assay mixtures included both thecommercial heart holoenzyme and its catalytic subunit. Myelin, thesource of the cAMP-independent protein kinase C family, was prepared bythe procedures of Wu et al. (Biochem. J., 209: 789-795 (1983)). Reactionmixtures (100 μl) also contained 1.0 μmole MgCl₂, 1.0 μmoledithiothreital, 200 μg histones as phosphorylation substrates. In thecase of the holoenzyme of heart protein kinase A 1.0 nmole cAMP was alsoadded. Urea (10 μmole) was routinely included following demonstrationthat low urea concentrations prevent polymerizations of the peptides.

Following peptide-enzyme interactions for varying periods the kinasereactions were initiated by additions of 22.5 nmole gamma-³²P-ATP (30mCi/mmole). After periodic incubations at 37° C., reactions were stoppedby additions of 0.02 ml glacial acetic acid. Aliquots (10 μl) of eachassay mixture were spotted on instant thin layer chromatography strips.The phosphorylated substrates were then separated from free ATP andmeasured for radioactive content by the method of Huang and Robinson(Anal. Biochem. 72: 593-599 (1976)). FIG. 1 demonstrates effects ofvarying concentrations of both the alpha-neurotoxin and its nontoxic,oxidized derivative on the activity of the catalytic subunit of heartprotein kinase. FIG. 2 is a Lineweaver-Burke analysis of the rates ofinhibition of the catalytic sub-unit of the heart kinase by the oxidizedpeptide at varying substrate concentrations. FIG. 3 depicts inhibitionof the myelin kinase isoforms by the oxidized peptide. Under those samereaction conditions, but without the presence of histones as substrate,the peptides exhibited no uptake of radioactive phosphate. Thus, kinaseinhibition is not due to competition by the peptides for phosphorylationsites on the protein substrate.

Confirmation of peptide inhibition of protein kinase A utilized theelectrophoretic PK-A assay method of Lutz (Lutz, et al., Anal. Biochem.220: 268-274 (1994)). Reaction mixtures in 50 microliters of 50 mM HEPESbuffer, pH 7.4, (1 mM with respect to EGTA) contained 2-4 units of thecatalytic subunit of heart protein kinase A, 8 nmole of kemptide (thePKA substrate), 500 nmole MgCl₂, and varying quantities of the oxidizedform of PANAVIRA®. Kinase reactions were initiated on additions of 10nmole ATP. After varying incubation times at 37 degrees C., thereactions were terminated by heat at 93 degrees C. for 5 minutes. Fiftymicroliters of 0.4 M borate buffer, pH 9.0 (20% with respect toglycerol), were added to each reaction tube, followed by 50 microlitersof acetone containing 20 micrograms fluorescamine. After 20 minutes atroom temperature for derivatization of the residual kemptide and newlyformed kemptide-phosphate, aliquots of the mixtures (15 microliters)were placed in slots in electrophoretic gel plates composed of 0.8%agarose in 50 mM HEPES buffer at pH 8.0. Electrophoresis at 96 Vseparates the unlabeled and phosphorylated kemptides as, respectively,cathodal and anodal fluorescent bands. Quantitations were by relativefluorescence of the two bands as fractions of that of the respectivecombined bands.

Example 2 Malignant Melanoma

A 12 year-old cocker spaniel presented with a diagnosis of Phase IImalignant melanoma. Multiple lesions, malodorous and resembling bunchedgrapes, emanated from the medial and lateral aspects of themucocutaneous junction of the right face. The patient was in obviouspain with face swollen and inflamed. Within 24-48 hours followingtreatment with PANAVIRA® (i.e. the polypeptides prepared according tothe method set forth in U.S. Pat. No. 7,259,237) the dog becameanimated, with lesions less inflamed. After one month of daily PAMAVIRA®treatment the lesions resembled dried raisins surrounded by healthytissue. The tumors were easily removed, and peptide therapy wascontinued for an additional month. During the subsequent four years nolesions recurred. On eventual death of the dog from renal failure nodetectable tumors were found on autopsy.

The same protocol as described above was subsequently applied to threeother dogs with Phase II-III malignant melanomas. Therapeutic outcomeswere identical.

Of the three mechanisms of action enjoyed by PANAVIRA®, the combinedneurotropism and ability to inhibit protein kinases are responsible forthe cures of these animals. Melanocytes are derived from the embryologicneural crest, showing that the neurotropism is reflected in the clinicalresponses. Melanoma cells, particularly metastatic melanoma cells, are,like nerve cells, rich in receptors for nerve growth factor (NGF)(Fabricant et al., Proc. Natl. Acad. Sci., 74: 565-569 (1977)). However,disorders in protein kinase functions also occur in melanoma cells. Thatis indicated by presence of a serine kinase activity associated withCD63, a stage-specific melanoma antigen (Iida, J., et al., J. Transl.Med., 3: 42-(2005)). Also, the mitogen-activated protein kinase (MAP)genes are significantly up-regulated in metastatic melanoma cell lines(Nambiar, S, et al., Arch. Dermatology 141: 163-173 (2005)). Further,the MAP kinase itself can up-regulate genes involved in production ofthe vascular endothelial growth factor (VEGF) and tissue factor (TF),both substances associated with the angiogenesis that supports tumorproliferation (Arbiser, J. L., et al.). The protein kinase assay systemthat documents peptide inhibition of the myelin protein kinase C,described above, involves inhibition of all isoforms of that enzymepresent in the myelin preparations. Selzer et al., (Melanoma Research,12: 201-209 (2002)) noted that, among the isoforms of the PKC family,the PKC-iota (PKC-i) isoform, was present in all melanoma tumor lysates,melanoma cell lines, and spontaneously transformed melanoma cells, butabsent in normal melanocytes. Since some protein kinases are knownexpression products of oncogenes, substantial attention falls oninhibitors of the kinases. For example, components of the anthrax toxininhibit the MAP kinase of melanoma cells. PANAVIRA® and its subset ofanalogues offer similar, safe responses.

Example 3 Canine Influenza

A two year-old racing greyhound, partially recovered from a severe boutof flu-like symptoms, retained loud rales in both lungs and continued ina debilitated condition in spite of continuing antibiotics andsupportive treatment of various kinds. Canine influenza was diagnosedvia blood tests at the Cornell Veterinary Diagnostic Laboratory.PANAVIRA® treatment was then instituted every twelve hours. Recovery wasevident within 3-4 days, and treatment was continued for 3-4 months.That dog then won first place in his class approximately six monthslater. During the same time period canine influenza was diagnosed in twodifferent litters of greyhound pups at the same farm. Twice dailytreatments with PANAVIRA® resulted in complete cessation of symptoms inall animals within 4-6 days, and with no recurrences.

Those results support clinical efficacy of PANAVIRA® in canineinfluenza. Canine influenza represents a recent expansion of the hostrange of influenza viruses. Common hosts for that virus include fowl,pigs, horses, and humans. Recently Crawford and associates isolatedinfluenza viruses from an outbreak among racing greyhounds in Florida(Science, 310: 482-485 (2005)). Analyses found the virus structureclosely related that of the H3N8 equine influenza virus. Identificationof the same virus among greyhounds in another geographic region, andsimilar findings among the general canine population, suggests efficienttransmission among the canine population (Yoon, et al., EmergingInfectious Diseases, 11: (1005)). The ability of PANAVIRA® to inhibitthe neuraminidases of myxoviruses (U.S. Pat. No. 7,259,237 B1) bestexplains the responses seen in influenza cases described above.

Example 4 Tourettes' Syndrome

A 33 year old male patient experienced the onset of the TourreteSyndrome during the 7-10 year age range. He was treated with Haldoluntil age 20 at which time that therapy was discontinued because of drugrelated limitations in life style and constant lethargy. At age 33 hewas treated with 0.2 ml of PANAVIRA® intramuscularly. A transitorymuscular tingling was noted approximately 45 minutes post injection.Twenty-four hours later the dose was doubled to 0.4 ml I.M. Forty-fiveminutes after that injection the patient experienced complete cessationof all evidence of Tourette's Syndrome with normalcy maintained at thesame daily dosage levels from that day forward. Normalcy ischaracterized by freedom from tics and speech impediments, and byuninterrupted ability to formulate sentences without hesitation.

Of the three mechanisms of peptide action described above theneurotropic property of the peptide and its ability to modulate proteinkinase responses to nerve cell stimuli provide the clinical response inTourette's Syndrome.

Example 5 Pain Associated Conditions

In the course of treatments of animals with varying clinical conditions,the dramatic analgesic effects of the same subset of peptides thatexhibit antiviral effects were immediately identified. Regardless of thedirect causes of pain, with few exceptions, rapid, dramatic relief ofpain is maintained with daily treatments.

Exemplifying alleviation of cancer pain was an eighteeen-month-oldGerman Shepherd presenting with a non-weight-bearing front leg due toosteosarcoma. Thirty to forty minutes after an initial peptide treatmenthe was observed running on all four legs with normal gait. Dailyinjections maintained that condition until death six months later.

Neurologic pain and/or neurologic deficits present major uses for thepeptides apart from their antiviral properties. Some examples includeboth animal and human conditions. A six-year-old dachshund presentedwith complete paralysis from the mid-lumbar area with dragging of bothrear legs. A tentative diagnosis of prolapsed disk syndrome was made andan initial treatment with the peptide was administered. At a follow-upvisit the next day the patient was able to stand, but could not walk.Follow-up injections were given for three days at which time the animalwas able to run and play normally.

Three dogs exemplify peptide treatment of stroke with flaccid paralysis.The first dog presented within hours of the stroke event. He was able tostand with help 24 hours after a first peptide injection, and walkednormally following the third treatment. The second dog, presented twodays after the stroke event, exhibited both paralysis and a severe headtilt. Four days of daily peptide injections enabled the animal to walk.Continued improvements followed daily injections. After one month ofdaily therapy the head tilt was resolved, thus completing recovery. Thethird dog was presented approximately two weeks following onset ofstroke symptoms. Peptide effects were not as pronounced as in the priorcases. The animal was able to stand up and walk only with help from theowner. No visible responses were noted during three weeks of dailyinjections. Suddenly, he then struggled to his feet without help. Theresponse from that point onward was dramatic with normal ambulationafter seven more daily injections.

An elderly man was given peptide injections for uncontrolled painassociated with a stroke twenty-seven years previously. Pain relief camewithin thirty minutes of the first dose, and was maintained by dailydoses for six months.

A fifty-two year old female underwent a hysterectomy for removal of alarge fibroid. Seven weeks later she developed a bladder fistula, whichwas corrected surgically. Following those surgeries she slept 13-14hours per day and was continually weak and tired. Physicianconsultations followed over a two-year period with the ultimatediagnosis of chronic fatigue syndrome and onset of fibromyalgia. Withdietary changes and a limited exercise regimen she gradually improvedbut continued to need 10 hours of sleep each night with restricted dailyactivity. After her first day of peptide treatment her leg strengthbegan to return. She continued daily injections for six months at whichtime she had returned to her normal pain-free life-style.

Of the three mechanisms of peptide action described above theneurotropic property of the peptides and their abilities to modulateprotein kinase responses to nerve cell stimuli provide the responses topain described here.

In summary, one polypeptide (and its subset of chemically modifiedderivatives described in U.S. Pat. No. 7,259,237) express three distinctmechanisms of action; namely, neurotropism, viral neuraminidaseinhibition, and mammalian protein kinase inhibition. Those mechanismshelp understand the broad range of therapeutic and prophylactic effectsdescribed herein. Positive effects are seen in multiple clinicalconditions such as in malignant melanoma, canine influenza A infection,Tourette's Syndrome, and analgesic effects in animals and humans. Thoseclinical applications are in addition to the effective therapies forfeline leukemia and feline immunodeficiency viral infections asdocumented in U.S. Pat. No. 7,259,237.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.Full citations for the publications are listed below. The disclosures ofthese publications and patents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

1. A method of inhibiting protein kinases, including the steps ofadministering polypeptides derived from alpha-neurotoxin, and inhibitingprotein kinases.
 2. (canceled)
 3. The method of claim 1, wherein saidadministrating step is further defined as injecting the polypeptides. 4.(canceled)
 5. The method of claim 1, wherein the polypeptides areprepared by methods chosen from the group consisting of oxidation, andreduction and alkylation of disulfide bonds of alpha-neurotoxin. 6.(canceled)
 7. The method of claim 22, further including the step oftreating cancer. 8.-12. (canceled)
 13. The method of claim 26, furtherincluding the step of treating Tourette's syndrome.
 14. The method ofclaim 13, wherein said treatment of Tourette's syndrome is furtherdefined as removing symptoms chosen from the group consisting of ticsand speech impediments.
 15. The method of claim 1, wherein saidinhibiting protein kinases step is further defined as modulating proteinkinase responses to nerve cell stimuli. 16.-19. (canceled)
 20. Themethod of claim 1, wherein said inhibiting protein kinases step isfurther defined as inhibiting cAMP-dependent protein kinases.
 21. Themethod of claim 1, wherein said inhibiting protein kinase step isfurther defined as inhibiting cAMP independent protein kinases includingthe inhibition of PKC enzymes.
 22. The method of claim 21 whereininhibited PKC enzymes include the Mitogen Activated Protein Kinases(MAPK) with inhibition of down stream regulation.
 23. The method ofclaim 22 wherein said MAPK enzymes and down regulation pathways areinhibited, includes a step in treating specific cancers includingmalignant melanoma, breast cancer, lung cancer, bowel cancer andprostate cancer.
 24. The method of claim 21, wherein the cancer ismalignant melanoma, and said inhibiting protein kinases step is furtherdefined as down-regulating MAP kinases, serine kinases, and threoninekinases.
 25. The method of claim 21, wherein said PKC enzymes and downregulation is inhibited, including treatments for retroviruses andlentiviruses including human immunodeficiency virus (HIV), FelineImmunodeficiency Virus, Feline Leukemia Virus and Equine InfectiousAnemia Virus.
 26. The method of claim 22, further including the step ofdown-regulating production of vascular endothelial growth factor (VEGF),other tissue factors (TF) and cytokines.